Role of mammalian target of rapamycin in the formation and progression of retinopathy of prematurity-like vascular abnormalities in neonatal rats.

Retinopathy of prematurity (ROP), a retinal disease that can occur in premature infants, can lead to severe visual impairment. In this study, we examined the preventive and therapeutic effects of mammalian target of rapamycin complex 1 (mTORC1) inhibition on abnormal retinal blood vessels in a rat model of ROP. To induce ROP-like vascular abnormalities, rats were subcutaneously treated with KRN633, an inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinase, on postnatal day 7 (P7) and P8. KRN633-treated (ROP) rats were treated subcutaneously with the mTORC1 inhibitor rapamycin according to preventive and therapeutic protocols, i.e., from P11 to P13 (P11-P13) and from P14 to P20 (P14-P20), respectively. To compare with the effects of VEGF inhibition, KRN633 was administered according to similar protocols. Changes in retinal vasculature, phosphorylated ribosomal protein S6 (pS6), a downstream indicator of mTORC1 activity, and the proliferative status of vascular cells were evaluated at P14 and P21 using immunohistochemistry. Rapamycin treatment from P11 to P13 prevented increases in arteriolar tortuosity, capillary density, and the number of proliferating vascular cells, and eliminated pS6 immunoreactivity in ROP rats. KRN633 treatment at P11 and P12 (P11/P12) also prevented the appearance of ROP-like retinal blood vessels. Rapamycin treatment from P14 to P20 failed to attenuate arteriolar tortuosity but prevented increases in capillary density and proliferating vascular cell number at the vascular front, but not at the central zone. KRN633 treatment from P14 to P20 significantly reduced abnormalities in the retinal vasculature; however, the effects were inferior to those of KRN633 treatment on P11/P12. These results suggest that activation of the mTORC1 pathway in proliferating endothelial cells contributes to the appearance and progression of ROP-like retinal blood vessels. Therefore, inhibition of mTORC1 may be a promising approach for selectively targeting abnormal retinal blood vessels in ROP.

The process of methylglyoxal-induced retinal capillary endothelial cell degeneration in rats.

Methylglyoxal, a highly reactive dicarbonyl compound, is increased and accumulated in patients with diabetic mellitus. Methylglyoxal forms advanced glycation end products (AGE), contributing to the pathogenesis of diabetic complications, including diabetic retinopathy. Recent studies have shown that methylglyoxal induces diabetic retinopathy-like abnormalities in retinal vasculature. In this study, we investigated the processes and mechanisms of methylglyoxal-induced retinal capillary endothelial cell degeneration in rats. Morphological changes in vascular components (endothelial cells, pericytes, and basement membranes) were assessed in the retinas 2, 7, and 14 days after intravitreal injection of methylglyoxal. Intravitreal methylglyoxal injection induced retinal capillary endothelial cell degeneration in a dose- and time-dependent manner. Changes in the shape and distribution of pericytes occurred before the initiation of capillary regression in the retinas of methylglyoxal-injected eyes. The receptor for AGEs (RAGEs) antagonist FPS-ZM1, and the matrix metalloproteinase (MMP) inhibitor GM6001 significantly attenuated methylglyoxal-induced capillary endothelial cell degeneration. FPS-ZM1 failed to prevent pathological changes in pericytes in methylglyoxal-injected eyes. In situ zymography revealed that MMP activity was enhanced at sites of blood vessels with reduced pericyte coverage in methylglyoxal-injected eyes. These results suggest that intravitreal methylglyoxal injection induces pathological changes in pericytes before the initiation of capillary endothelial cell degeneration via an AGE-RAGE-independent pathway. The capillary endothelial cell degeneration is mediated by activating the AGE-RAGE pathway and increasing MMP activity in endothelial cells by impairing pericyte function in the retina.

Tumor necrosis factor-α and matrix metalloproteinase-9 cooperatively exacerbate neurovascular degeneration in the neonatal rat retina.

Matrix metalloproteinases (MMPs) and tumor necrosis factor (TNF)-α contribute to the pathogenesis of several ocular diseases. Previous studies have shown that MMP-9 activation plays an important role in capillary degeneration in injured retinas. In this study, we aimed to determine the roles of TNF-α in capillary degeneration and MMP-9 activation in the injured retina. In rats, retinal injury was induced by intravitreal injection of N-methyl-D-aspartic acid (NMDA, 200 nmol) at postnatal day 7. We examined (1) the effects of blocking MMP-9 and TNF-α signaling pathway on capillary degeneration, (2) changes in protein levels and distribution of MMP-9 and TNF-α, and (3) the interaction between MMP-9 and TNF-α in regulating the expression level of each protein in retinas of NMDA-injected eyes. Intravitreal injection of GM6001, an MMP inhibitor, or TNF-α neutralizing antibody (anti-TNF-α Ab) attenuated capillary degeneration in retinas of NMDA-injected eyes. Protein levels of TNF-α increased 2 h after NMDA injection, whereas those of MMP-9 increased 4 h after the injection. Anti-TNF-α Ab suppressed activation of MMP-9 in retinas of NMDA-injected eyes, whereas GM6001 diminished the TNF-α protein expression. Incubation of recombinant TNF-α with supernatants of homogenized retina increased protein levels and activity of MMP-9. These results suggest that TNF-α and MMP-9 collaboratively increase their expression levels in the retina following neurodegeneration, thus leading to retinal capillary degeneration. The cooperative interaction between MMP-9 and TNF-α could be involved in the exacerbation of retinal neurovascular degeneration.

Pharmacological inhibition of Na+/K+-ATPase induces neurovascular degeneration and glial cell alteration in the rat retina.

Na+/K+-ATPase (NKA) plays an important role in ion homeostasis and neurotransmitter uptake. In the retina, multidirectional communications among neurons, glia, and blood vessels (that is, neuro-glio-vascular interaction) are crucial for maintaining tissue homeostasis. We investigated the role of NKA in the elements of neuro-glio-vascular unit in neonatal and adult rat retinas. Male Sprague-Dawley rats (1- and 8-week-old) were injected intravitreally with ouabain (20 nmol/eye), an inhibitor of NKA. Morphological changes in retinal neurons, glia, and blood vessels were examined. The intravitreal injection of ouabain decreased the number of cells in the ganglion cell layer, as well as the thicknesses of the inner plexiform and inner nuclear layers in neonatal and adult rats compared to age-matched controls. The ouabain-induced neuronal cell damage was partially prevented by D-(-)-2-amino-5-phosphonopentanoic acid, an antagonist of N-methyl-D-aspartic acid receptors. In the deep retinal vascular plexus of the ouabain-injected eyes, angiogenesis was delayed in neonatal rats, whereas capillary degeneration occurred in adult rats. The immunoreactivity of glutamine synthetase and vascular endothelial growth factor (VEGF) decreased in the retinas of neonatal and adult rats injected intravitreally with ouabain. The immunoreactivity of glial fibrillary acidic protein was enhanced in the retinas of ouabain-injected adult eyes. After the ouabain injection, CD45-positive leukocytes and Iba1-positive microglia increased in the inner retinal layer of neonatal rats, whereas they increased in the middle retinal layer of adult rats. These results suggest that the inhibition of NKA induces the degeneration of neuronal and vascular cells and alteration of glial cells in both neonatal and adult retinas. In addition to the direct effects of NKA inhibition, the disturbance of retinal glutamate metabolism and decreased VEGF expression may contribute to neurovascular degeneration. The activity of NKA is crucial for maintaining elements of neuro-glio-vascular unit in the retina.

Pharmacological depletion of retinal neurons prevents vertical angiogenic sprouting without affecting the superficial vascular plexus.

BACKGROUND: In mice, a tri-layered (superficial, intermediate, and deep) vascular structure is formed in the retina during the third postnatal week. Short-term treatment of newborn mice with vascular endothelial growth factor (VEGF) receptor inhibitors delays the formation of superficial vascular plexus and this allows us to investigate the developmental process of superficial and deep vascular plexuses at the same time. Using this model, we examined the effect of pharmacological depletion of retinal neurons on the formation of superficial and deep vascular plexuses. RESULTS: Neuronal cell loss induced by an intravitreal injection of N-methyl-d-aspartic acid on postnatal day (P) 8 delayed vascular development in the deep layer but not in the superficial layer in mice treated with KRN633, a VEGF receptor inhibitor, on P0 and P1. In KRN633-treated mice, neuronal cell loss decreased the number of vertical sprouts originating from the superficial plexus without affecting the number of angiogenic sprouts growing in front. Neuronal cell loss did not impair networks of fibronectin and astrocytes in the superficial layer. CONCLUSIONS: Our results suggest that inner retinal neurons play a crucial role in forming the deep vascular plexus by directing the sprouts from the superficial blood vessels to the deep layer.

Impairment of endothelium-dependent vasodilator function of retinal blood vessels in adult rats with a history of retinopathy of prematurity.

Retinopathy of prematurity (ROP) is a proliferative retinal vascular disease, initiated by delayed retinal vascular growth after premature birth. In the majority of cases, ROP resolves spontaneously; however, a history of ROP may increase the risk of long-term visual problems. In this study, we evaluated the endothelial function of retinal blood vessels in adult rats with a history of ROP. ROP was induced in rats by subcutaneous injection of a vascular endothelial growth factor receptor tyrosine kinase inhibitor (KRN633) on postnatal day (P) 7 and P8. On P56, vasodilator responses to acetylcholine, GSK1016790A (an activator of transient receptor potential vanilloid 4 channels), NOR3 (a nitric oxide [NO] donor), and salbutamol (a ß2-adrenoceptor agonist) were assessed. Compared to age-matched controls, retinal vasodilator responses to acetylcholine and GSK1016790A were attenuated in P56 rats with a history of ROP. No attenuation of acetylcholine-induced retinal vasodilator response was observed under inhibition of NO synthase. Retinal vasodilator responses to NOR3 and salbutamol were unaffected. These results suggest that the production of and/or release of NO is impaired in retinal blood vessels in adult rats with a history of ROP. A history of ROP might increase the risk of impaired retinal circulation in adulthood.

Metformin Protects against NMDA-Induced Retinal Injury through the MEK/ERK Signaling Pathway in Rats.

Metformin, an anti-hyperglycemic drug of the biguanide class, exerts positive effects in several non-diabetes-related diseases. In this study, we aimed to examine the protective effects of metformin against N-methyl-D-aspartic acid (NMDA)-induced excitotoxic retinal damage in rats and determine the mechanisms of its protective effects. Male Sprague-Dawley rats (7 to 9 weeks old) were used in this study. Following intravitreal injection of NMDA (200 nmol/eye), the number of neuronal cells in the ganglion cell layer and parvalbumin-positive amacrine cells decreased, whereas the number of CD45-positive leukocytes and Iba1-positive microglia increased. Metformin attenuated these NMDA-induced responses. The neuroprotective effect of metformin was abolished by compound C, an inhibitor of AMP-activated protein kinase (AMPK). The AMPK activator, AICAR, exerted a neuroprotective effect in NMDA-induced retinal injury. The MEK1/2 inhibitor, U0126, reduced the neuroprotective effect of metformin. These results suggest that metformin protects against NMDA-induced retinal neurotoxicity through activation of the AMPK and MEK/extracellular signal-regulated kinase (ERK) signaling pathways. This neuroprotective effect could be partially attributable to the inhibitory effects on inflammatory responses.

Changes in components of the neurovascular unit in the retina in a rat model of retinopathy of prematurity.

An impairment of cellular interactions between the elements of the neurovascular unit contributes to the onset and/or progression of retinal diseases. The present study aims to examine how elements of the neurovascular unit are altered in a rat model of retinopathy of prematurity (ROP). Neonatal rats were treated subcutaneously with the vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor KRN633 (10 mg/kg) on postnatal day (P) 7 and P8 to induce ROP. Morphological assessments were performed of blood vessels, astrocytes and neuronal cells in the retina. Aggressive angiogenesis, tortuous arteries and enlarged veins were observed in the retinal vasculature of KRN633-treated (ROP) rats from P14 to P28, compared to age-matched control (vehicle-treated) animals. Morphological abnormalities in the retinal vasculature showed a tendency toward spontaneous recovery from P28 to P35 in ROP rats. Immunofluorescence staining for glial fibrillary acidic protein and Pax2 (astrocyte markers) revealed that morphological changes to and a reduction in the number of astrocytes occurred in ROP rats. The developmental cell death was slightly accelerated in ROP rats; however, no visible changes in the morphology of retinal layers were observed on P35. The abnormalities in astrocytes might contribute, at least in part, to the formation of abnormal retinal blood vessels and the pathogenesis of ROP.

The process of revascularization in the neonatal mouse retina following short-term blockade of vascular endothelial growth factor receptors.

Misdirected vascular growth frequently occurs in the neovascular diseases in the retina. However, the mechanisms are still not fully understood. In the present study, we created capillary-free zones in the central and peripheral retinas in neonatal mice by pharmacological blockade of vascular endothelial growth factor (VEGF) signaling. Using this model, we investigated the process and mechanisms of revascularization in the central and peripheral avascular areas. After the completion of a 2-day treatment with the VEGF receptor tyrosine kinase inhibitor KRN633 on postnatal day (P) 4 and P5, revascularization started on P8 in the central avascular area where capillaries had been dropped out. The expression levels of VEGF were higher in the peripheral than in the central avascular area. However, the expansion of the vasculature in the peripheral avascular retina remained suppressed until revascularization had been completed in the central avascular area. Additionally, we found disorganized endothelial cell division, misdirected blood vessels with irregular diameters, and abnormal fibronectin networks at the border of the vascular front and the avascular retina. In the central avascular area, a slight amount of fibronectin as non-vascular component re-formed to provide a scaffold for revascularization. Mechanistic analysis revealed that higher levels of VEGF attenuated the migratory response of endothelial cells without decreasing the proliferative activity. These results suggest that the presence of concentration range of VEGF, which enhances both migration and proliferation of the endothelial cells, and the structurally normal fibronectin network contribute to determine the proper direction of angiogenesis.

Abnormal Vascular Phenotypes Associated with the Timing of Interruption of Retinal Vascular Development in Rats.

Pathological angiogenesis is a leading cause of blindness in several retinal diseases. The key driving factor inducing pathological angiogenesis is the pronounced hypoxia leading to a marked, increased production of vascular endothelial growth factor (VEGF). The aim of this study was to determine whether the abnormal vascular growth occurs in a manner dependent on the degree of the vascular defects. Vascular defects of two different degrees were created in the retina by subcutaneously treating neonatal rats with the VEGF receptor (VEGFR) tyrosine kinase inhibitor KRN633 on postnatal day (P) 4 and P5 (P4/5) or P7 and P8 (P7/8). The structure of the retinal vasculature changes was examined immunohistochemically. Prevention of vascular growth and regression of some preformed capillaries were observed on the next day, after completion of each treatment (i.e., P6 and P9). The vascular regrowth occurred as a result of eliminating the inhibitory effect on the VEGFR signaling pathway. KRN633 (P4/5)-treated rats exhibited a retinal vasculature with aggressive intravitreal neovascularization on P21. On the other hand, the appearance of tortuous arteries is a representative vascular pathological feature in retinas of KRN633 (P7/8)-treated groups. These results suggest that an interruption of the retinal vascular development at different time points induces different vascular pathological features in the retina. Pharmacological agents targeting the VEGF signaling pathway are useful for creating an abnormal retinal vasculature with various pathological features in order to evaluate the efficacy of anti-angiogenic compounds.

Involvement of matrix metalloproteinases in capillary degeneration following NMDA-induced neurotoxicity in the neonatal rat retina.

Interactions between neuronal cells and vascular cells in the retina are critical for maintaining retinal tissue homeostasis. Impairment of cellular interactions contributes to development and progression of retinal diseases. Previous studies demonstrated that neuronal cell damage leads to capillary degeneration in an N-methyl-D-aspartic acid (NMDA)-induced retinal degeneration model. However, the mechanisms underlying this phenomenon are not fully understood. In this study, we examined the possible role of matrix metalloproteinase (MMP)-9 in neuronal cell loss and capillary degeneration in NMDA-treated retinas of neonatal rats. Intravitreal injection of NMDA (50 or 200 nmol) was performed on postnatal day (P) 7 and morphological changes in retinal neurons and vasculature were examined on P14. The MMP inhibitor CP101537 (100 nmol) or vehicle (dimethyl sulfoxide) was intravitreally injected simultaneously with, or 2 days after, NMDA injection. CP101537 protected against neurovascular degeneration in a time-dependent manner as follows: 1) simultaneous injection of CP101537 with NMDA prevented morphological changes in retinal neurons induced by NMDA (50 nmol); and 2) reduction in capillary density and number of vertical sprouts induced by NMDA (200 nmol) was prevented when CP101537 was injected 2 days after NMDA injection. Gelatin zymography and western blot analyses indicated that activity and protein levels of MMP-9 were enhanced from 4 h to 2 days after NMDA injection. Increased activity and protein levels of MMP-9 were suppressed by MMP inhibitors (CP101537 and GM6001). In situ zymography revealed that MMP activity was enhanced throughout the retinal vasculature in NMDA-treated retinas. These results indicate that MMP-9 plays an important role in neurovascular degeneration in the injured retina. Inhibition of MMP-9 may be an effective strategy for preventing and reducing neurovascular degeneration.

Cellular Mechanisms of Angiogenesis in Neonatal Rat Models of Retinal Neurodegeneration.

Νeuronal and glial cells play an important role in the development of vasculature in the retina. In this study, we investigated whether re-vascularization occurs in retinal neurodegenerative injury models. To induce retinal injury, N-methyl-D-aspartic acid (NMDA, 200 nmol) or kainic acid (KA, 20 nmol) was injected into the vitreous chamber of the eye on postnatal day (P)7. Morphological changes in retinal neurons and vasculature were assessed on P14, P21, and P35. Prevention of vascular growth and regression of some capillaries were observed on P14 in retinas of NMDA- and KA-treated eyes. However, vascular growth and re-vascularization started on P21, and the retinal vascular network was established by P35 in retinas with neurodegenerative injuries. The re-vascularization was suppressed by a two-day treatment with KRN633, an inhibitor of VEGF receptor tyrosine kinase, on P21 and P22. Astrocytes and Müller cells expressed vascular endothelial growth factor (VEGF), and the distribution pattern of VEGF was almost the same between the control and the NMDA-induced retinal neurodegenerative injury model, except for the difference in the thickness of the inner retinal layer. During re-vascularization, angiogenic sprouts from pre-existing blood vessels were present along the network of fibronectins formed by astrocytes. These results suggest that glial cells contribute to angiogenesis in neonatal rat models of retinal neurodegeneration.

Role of Neuron⁻Glia Signaling in Regulation of Retinal Vascular Tone in Rats.

The interactions between neuronal, glial, and vascular cells play a key role in regulating blood flow in the retina. In the present study, we examined the role of the interactions between neuronal and glial cells in regulating the retinal vascular tone in rats upon stimulation of retinal neuronal cells by intravitreal injection of N-methyl-d-aspartic acid (NMDA). The retinal vascular response was assessed by measuring the diameter of the retinal arterioles in the in vivo fundus images. Intravitreal injection of NMDA produced retinal vasodilation that was significantly diminished following the pharmacological inhibition of nitric oxide (NO) synthase (nNOS), loss of inner retinal neurons, or intravitreal injection of glial toxins. Immunohistochemistry revealed the expression of nNOS in ganglion and calretinin-positive amacrine cells. Moreover, glial toxins significantly prevented the retinal vasodilator response induced by intravitreal injection of NOR3, an NO donor. Mechanistic analysis revealed that NO enhanced the production of vasodilatory prostanoids and epoxyeicosatrienoic acids in glial cells in a ryanodine receptor type 1-dependent manner, subsequently inducing the retinal vasodilator response. These results suggest that the NO released from stimulated neuronal cells acts as a key messenger in neuron-glia signaling, thereby causing neuronal activity-dependent and glial cell-mediated vasodilation in the retina.

Transient phenotypic changes in endothelial cells and pericytes in neonatal mouse retina following short-term blockade of vascular endothelial growth factor receptors.

BACKGROUND: A short-term interruption of vascular development causes structural abnormalities in retinal vasculature. However, the detailed changes in vascular components (endothelial cells, pericytes, and basement membranes) remain to be fully determined. The present study aimed to provide a detailed description of morphological changes in vascular components following a short-term interruption of retinal vascular development in mice. RESULTS: Two-day treatment of neonatal mice with the vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor KRN633 (10 mg/kg, subcutaneously) on postnatal day (P)0 and P1 (P0/1) and P4 and P5 (P4/5) induced different degrees and patterns of impairment of retinal vascular development. Three days after completion of the treatment, the delayed radial vascular growth occurred in P0/1 group mice, whereas in P4/5 group mice, revascularization preferentially occurred in the central avascular area, and radial vascular growth remained suppressed by P10. Differences in α-smooth muscle actin expression in pericytes were noted in the processes between normal vascular formation and vascular regrowth. The changes in vascular cells were associated with the hypoxia-induced enhancement of VEGF expression in the superficial retinal layer. CONCLUSIONS: These findings suggest that the phenotype of vascular cells is altered following a short-term interruption of vascular development in the retina. Developmental Dynamics 247:699-711, 2018. © 2017 Wiley Periodicals, Inc.

Establishment of an abnormal vascular patterning model in the mouse retina.

Abnormalities in retinal blood vessels and neuronal function persist in eyes undergoing retinopathy of prematurity. In this study, we examined morphological and functional changes in retinal blood vessels and neurons in mice that had undergone short-term interruption of retinal vascular development through inhibition of vascular endothelial growth factor (VEGF) signaling. In mice treated with the VEGF receptor tyrosine kinase inhibitor KRN633 on postnatal day (P) 0 and 1, the vascular density in the retinal surface increased by P12, but development of deep retinal vascular plexus and choroidal vasculature was delayed until P14. Overall retinal morphology was mostly normal in KRN633-treated mice during the observation period (∼P28), with the exception of P8 and P14. On P28, abnormalities in retinal vascular patterns were evident, but electroretinogram and retinal blood perfusion were within the normal range. Abnormal architecture of retinal vasculature disturbs retinal hemodynamics; therefore, mice treated postnatally with VEGF receptor inhibitors could serve as an animal model for studying the regulatory mechanism of local retinal blood flow and the effect of persistent abnormal retinal vascular patterns on the risk of onset of retinal ischemia.

Anti-angiogenic effects of valproic acid in a mouse model of oxygen-induced retinopathy.

Pathological retinal angiogenesis contributes to the pathogenesis of several ocular diseases. Valproic acid, a widely used antiepileptic drug, exerts anti-angiogenic effects by inhibiting histone deacetylase (HDAC). Herein, we investigated the effects of valproic acid and vorinostat, a HDAC inhibitor, on pathological retinal angiogenesis in mice with oxygen-induced retinopathy (OIR). OIR was induced in neonatal mice by exposure to 80% oxygen from postnatal day (P) 7 to P10 and to atmospheric oxygen from P10 to P15. Mice were subcutaneously injected with valproic acid, vorinostat, or vehicle once a day from P10 to P14. At P15, retinal neovascular tufts and vascular growth in the central avascular zone were observed in mice with OIR. Additionally, immunoreactivity for phosphorylated ribosomal protein S6 (pS6), an indicator of mammalian target of rapamycin (mTOR) activity, was detected in the neovascular tufts. Both valproic acid and vorinostat reduced the formation of retinal neovascular tuft without affecting vascular growth in the central avascular zone. Valproic acid reduced the pS6 immunoreactivity in neovascular tufts. Given that vascular endothelial growth factor (VEGF) activates mTOR-dependent pathways in proliferating endothelial cells of the neonatal mouse retina, these results suggest that valproic acid suppresses pathological retinal angiogenesis by interrupting VEGF-mTOR pathways.

A delay in vascularization induces abnormal astrocyte proliferation and migration in the mouse retina.

BACKGROUND: Astrocytes migrate into the retina through the optic nerve head by means of the axons of retinal ganglion cells, and spread radially toward the peripheral retina. Endothelial cells migrate along the astrocyte cellular network to form the retinal surface vasculature. Here, we examined the effects of a delay in retinal vascularization on the migration and proliferation status of astrocytes in mice. RESULTS: A dose-dependent delay in retinal vascularization was observed in mice that had been treated with KRN633 (1-10 mg/kg), a VEGF receptor inhibitor, on the day of birth and on the following day. Delayed vascularization resulted in a delay in the astrocyte network formation, and an increase in astrocyte number in the optic nerve head and the vascular front. The increase in the number of astrocytes may be attributed to increased proliferation and delayed migration. These abnormalities in astrocyte behavior correlated with the degree of delay in retinal vascularization. The vascularization delay also led to retinal hypoxia, which subsequently stimulated VEGF leading to an increase in vascular density. CONCLUSIONS: These findings suggest that a delay in normal vascularization leads to abnormal astrocyte behavior, which results in the formation of abnormal astrocyte and endothelial cell networks in the mouse retina. Developmental Dynamics 246:186-200, 2017. © 2016 Wiley Periodicals, Inc.

Mammalian Target of Rapamycin (mTOR) as a Potential Therapeutic Target in Pathological Ocular Angiogenesis.

Pathological ocular angiogenesis is a causative factor of retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. Vascular endothelial growth factor (VEGF) plays an important role in pathological angiogenesis, and anti-VEGF agents have been used to treat the ocular diseases that are driven by pathological angiogenesis. However, adverse effects associated with the blockade of VEGF signaling, including impairments of normal retinal vascular growth and retinal function, were suggested. Therefore, the development of a safe, effective strategy to prevent pathological ocular angiogenesis is needed. Recent studies have demonstrated that inhibitors of the mammalian target of rapamycin (mTOR) target proliferating endothelial cells within the retinal vasculature. Here, we review the potential of targeting the mTOR pathway to treat pathological ocular angiogenesis.

Exposure to high-concentration oxygen in the neonatal period induces abnormal retinal vascular patterning in mice.

The interruption of vascular development could cause structural and functional abnormalities in tissues. We have previously reported that short-term treatment of newborn mice with vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitors induces abnormal retinal vascular growth and patterns. An exposure of neonatal mice to high-concentration oxygen disturbs normal retinal vascular development. The present study aimed to determine (1) whether vascular abnormalities are observed in the retina of newborn mice exposed to high concentrations of oxygen, and (2) how astrocyte network formation is affected following the exposure to hyperoxia. Newborn (postnatal day 0) mice were exposed to 75% oxygen for 48 or 96 hr. During hyperoxia exposure, VEGF expression decreased, and the onset of retinal vascularization was completely suppressed. After completion of the hyperoxic period, retinal vascularization occurred, but it was delayed in a hyperoxic exposure duration-dependent manner. In retinas of hyperoxia-exposed mice, dense capillary plexuses were found, and the number of arteries and veins decreased. The astrocyte network formation was slightly delayed under hyperoxic conditions, and the network became denser in retinas of mice with an episode of hyperoxia. Expression of VEGF levels in the avascular retina of mice that were exposed to hyperoxia was higher than that of control mice. These results suggest that short-term interruption of the onset of vascular development resulting from the reduction in VEGF signals induces abnormal vascular patterns in the mouse retina. The abnormalities in retinal astrocyte behavior might contribute to the formation of an abnormal retinal vascular growth.

Effects of pre- and post-natal treatment with KRN633, an inhibitor of vascular endothelial growth factor receptor tyrosine kinase, on retinal vascular development and patterning in mice.

The impaired function of angiogenic factors, including vascular endothelial growth factor (VEGF), during pregnancy is associated with preeclampsia and intrauterine growth restriction. To determine how the attenuation of VEGF signals during retinal vascular development affects retinal vascular growth and patterns, we examined the effects of pre- and post-natal treatment of mice with KRN633, a VEGF receptor tyrosine kinase inhibitor, on retinal vascular development and structure. Delays in retinal vascular development were observed in the pups of mother mice that were treated daily with KRN633 (5 mg/kg/day) from embryonic day 13.5 until the day of delivery. A more marked delay was seen in pups treated with the inhibitor (5 mg/kg/day) on the day of birth and on the following day. Pups treated postnatally with KRN633 showed abnormal retinal vascular patterns, such as highly dense capillary networks and decreased numbers of central arteries and veins. The high-density vascular networks in KRN633-treated pups showed a greater sensitivity to VEGF signaling inhibition than the normal vascular networks in vehicle-treated pups. Compared to vehicle-treated pups, more severe hypoxia and stronger VEGF mRNA expression were observed in avascular areas in KRN633-treated pups. These results suggest that a short-term loss of VEGF function at the earliest stages of vascular development suppresses vascular growth, leading to abnormal vascular patterning, at least in part via mechanisms involving VEGF in the mouse retina.